A.Retinol storage through esterification is reduced in breast cancer cells. We have hypothesized that essential nutrient deficiency, and in particular vitamin A deficiency, may be a tumor promoting agent in epithelial cells. We have shown that cultured human breast cancer cells are characterized by a marked reduction in their ability to synthesize the storage form of retinol, the retinyl esters, compared to cultured normal epithelial cells from the human mammary gland. This reduced ability was attributed to a reduced expression of the retinyl ester synthesizing enzyme, lecithin retinol acyl transferase (LRAT), in the MCF-7 cells as measured by Northern blot analysis of its 5 Kb transcript as well as by Western blot analysis, using polyclonal antibodies developed against the N- and the C-terminal domains of LRAT. A similar reduced expression was found in tumorigenic cell lines derived from mouse epidermal keratinocytes compared to normal keratinocytes. The polyclonal antibodies detected at least three LRAT isoforms, two as a doublet of molecular weight around 55 Kd and a third of about 25 Kd. This latter peptide corresponds to the expected size of the translated product, the other two isoforms appear to result from post-translational modifications of the shorter peptide. Since MCF-7 cells express a shorter (2.7 Kb) transcript than HMEC cells we will concentrate our efforts to clone and sequence this transcript and determine whether it represents a splice variant predominant in cancer cells. B.CYP2C39 regulates retinoic acid catabolism in mouse liver. Livers of the aryl hydrocarbon receptor (AHR) null mouse have abnormally high levels of retinoic acid (RA) and its precursor retinyl palmitate. Retinoid accumulation in these animals is thought to be responsible for the severe liver phenotype characterized by small size and fibrosis. The increased levels of hepatic RA are believed to be due to decreased metabolism of RA to 4-hydroxy retinoic acid, the principal route of RA catabolism in vertebrates. RA 4-hydroxylation in mammalian liver is NADPH-dependent and is inhibited by carbon monoxide, suggesting that it is mediated by cytochrome(s) P450. To identify the P450 isoforms involved in RA metabolism in mouse liver tissue, microsomes from AHR-null and wild-type mice were subjected to western blotting and probed with cross-reacting antibodies raised to P450s in other species. Signal intensity in western blots probed with two independently-derived anti-rat CYP2C6 antibodies correlated well with levels of RA 4-hydroxylation. Anti-2C6 antibody inhibited RA 4-hydroxylase activity in liver microsomes of wild-type mouse liver microsomes to the levels observed in AHR -/- mice. When used to screen a mouse liver cDNA expression library, this antibody exclusively recognized the murine P450 2C39. Catalytic assays of five recombinant mouse CYP2C P450s expressed in E. coli revealed that only CYP2C39 was competent for RA 4-hydroxylation. Consistent with the fact that hepatic RA-4 hydroxylation is not induced by TCDD treatment in this mouse line, CYP2C39 mRNA is expressed at only mildly elevated levels in wild-type animals treated with TCDD and is not reduced in the absence of the AHR. Thus, CYP2C39 protein but not mRNA is reduced in the AHR -/- mouse. These data suggest that CYP2C39 is an important enzyme catalyzing RA catabolism and ultimately controlling levels of RA in mouse liver.